Enterotoxigenic E. coli (ETEC) is one of the most frequent bacterial causes of diarrhea in children in developing countries. ETEC is also a major cause of traveler's diarrhea. Disease usually occurs following ingestion of ETEC and colonization of the intestinal mucosal surface. Colonization is facilitated by specific colonization factors (CF) located on the surface of the bacteria. Following colonization, the bacteria produce a heat-labile toxin (LT) and/or a heat stable toxin (ST) that trigger watery diarrhea. The objective of this invention is to develop an oral inactivated ETEC vaccine for use in children living in endemic areas as well as in Western travelers going to ETEC-endemic areas.
A previously developed oral vaccine consisting of inactivated ETEC bacteria and recombinant cholera toxin B-subunit (CTB) was shown to be safe and immunogenic in children living in endemic areas as well as in Western adults traveling to areas endemic for ETEC. This vaccine conferred some protection against moderate/severe diarrhea in adult travelers; however, the protective efficacy in children was not significant.
The inventors reviewed the information gained from the clinical studies of this 1st generation ETEC vaccine and concluded that a vaccine formulation containing increased amounts of CF antigens and with increased toxin neutralizing capacity should provide better protective immunity. One problem with simply increasing the amount of CF antigens is that too large number of cells in a vaccine dose result in adverse effects such as nausea and vomiting, in particular in infants.
Hence, a new 2nd generation, tetravalent ETEC vaccine was developed, containing four inactivated E. coli strains over-expressing, i.e. increased levels compared to clinical ETEC isolates, of the most prevalent CFs, i.e. CFA/I, CS3, CS5 and CS6, and a novel rLTB/CTB (LCTBA) hybrid protein “toxoid” with stronger immunogenicity against LT than the rCTB toxoid used in the previously studied formulation. A monovalent prototype of this 2nd generation vaccine was recently studied in a clinical phase I trial and was found to be immunogenic, safe and well tolerated by the subjects.
E. coli LT has both enterotoxic and adjuvant properties. However, the use of LT toxin as an oral adjuvant has been hampered by its enterotoxicity. To circumvent this problem, a double mutant LT (dmLT) toxoid, devoid of enterotoxicity but with retained adjuvant properties was recently developed. Safety of oral administration of a cGMP pilot lot of dmLT has been documented both in a preclinical GLP toxicology study and in an ongoing clinical study in the United States.
Here, the inventors demonstrate the efficacy a novel ETEC vaccine comprising dmLT adjuvant to enhance immuno responses.
The presently disclosed vaccine has particular benefits regard to its ability to effectively elicit immuno responses (in particular, to several CFs simultaneously) while keeping the amount of cells per unit dose sufficiently low to avoid adverse effects. Too large numbers of cells lead to adverse effects such as nausea and vomiting, in particular in infant subjects.
Certain aspects of the present invention have been disclosed by the inventors in an earlier academic publication (Svennerholm A M. From cholera to enterotoxigenic Escherichia coli (ETEC) vaccine development. Indian J Med Res. 2011 February; 133(2):188-96. Review).